Molecular machines are responsible for almost all central cellular functions and include the splicosome, RNA-induced interference complex, and ribosome. Biomedical researchers would like to follow as a function of time the position, composition, and structural and functional dynamics of single molecular machines inside living cells. Certain gold clusters are small, water soluble, brightly fluorescent, and photostable and have significant potential as labels for in vivo single molecule studies. However, gold clusters are not currently genetically encoded and it is not clear how to deliver them into the cytoplasm of living cells. Our hypothesis is that these two limitations can be resolved by specifically growing gold clusters inside living cells using scaffolded peptides. We will test this hypothesis with three specific research thrusts. 1. We will quantity the potential adverse effects of gold ions at the concentrations needed for intracellular cluster formation. HeLa tissue culture cells and zebrafish embryos/larvae will be exposed to chloroaurate for minutes to days. Potential adverse effects will be assessed by optical and electron microscopy. 2. We will quantify nonspecific labeling backgrounds and establish the ability of certain scaffolded peptides to direct in vivo cluster growth. In vivo cluster growth will be monitored with fluorescence and electron microscopy. 3. We will generate novel cluster nucleation peptides that perform well inside living cells. Scaffolded peptide libraries will be expressed in HeLa cells. Cells that become fluorescent upon gold exposure will be isolated by fluorescence-activated cell-sorting (FACS). Subsequent hit validation will involve epi-fluorescence, confocal, and electron microscopy. NIH PAR-07-234 has six aims;the proposed technology addresses PAR-07-234 objective 1, the development of new probes for light microscopy, objective 3, development of new classes of genetically encoded probes, and objective 4, genetically-encoded probes for electron and X-ray microscopy. As required by PAR-07-234, the proposed technology is unproven and novel, since 1) potential adverse effects have not been fully evaluated, especially not in a vertebrate model system, 2) it is not known whether fluorescent gold clusters can be specifically grown in vivo, 3) the ability of the technology to facilitate in vivo single molecule experiments and correlative optical, electron, and soft x-ray studies remains to be established, and 4) we will use FACS to identify scaffolded peptides that efficiently direct cluster growth in vivo. A multi-disciplinary team has been assembled to bring the needed expertise to these tasks. The three investigators are experts in single-molecule biophysics, optical and electron microscopy, development of new biomedical research tools, zebrafish physiology, and peptide-controlled synthesis of materials. They will be supported by two international experts in gene delivery, tissue engineering, and metal toxicology in zebrafish. The collaboration comprises researchers from Physics, Bioengineering, and Chemical Engineering (UC Berkeley), the Life Sciences and Physical Biosciences Depts. of Lawrence Berkeley Lab, and the Oregon State U. Dept. of Environmental and Molecular Toxicology. The proposed research will establish the feasibility of genetically encoded gold clusters. Such probes would represent a fundamentally new labeling technology differing from current methodologies by combining photostable fluorescence, genetic encoding, small size, and visibility in optical, electron, and X-ray microscopy ('multi-modality'). This research may greatly facilitate basic biomedical research by giving researchers a powerful new tool for watching how molecular machines work in their native context with high spatial and temporal resolution. Such measurements will reveal the roles of proteins within their tissue context and help to dissect molecular disease mechanisms, such as how mutant proteins disrupt interactions between transcription factors and impair the DNA replication machinery. The photostability of the probes may allow extended monitoring of individual proteins, enabling studies of gradual disease progression within cells and tissues. The brightness of the probes may permit early detection of cellular and tissue dysfunction in model systems. To ensure public health significance, much of the research will involve zebrafish, a vertebrate animal model system for an increasing number of human diseases.